Nanopores are now a well-established class of label-free sensors capable of detecting single molecules electrically. The technique relies on the application of a voltage across a nano-scale aperture in a thin, insulating membrane immersed in an ionic solution. Modulation of the resulting ionic current can be associated with the translocation of individual charged biomolecules such as DNA and proteins that are electrophoretically driven through the nanopore. These changes in conductance provide information about the length, size, charge and shape of translocating molecules. A variety of single-molecule studies, including DNA sequencing, protein detection and unfolding, single-molecule mass spectrometry and force spectroscopy make this technology particularly attractive.
Nanopores may be formed by incorporating proteinaceous pores in lipid bilayer membranes or fabricated in thin, solid-state membranes. The biological pores offer very low noise properties, but the high fragility of the conventionally used lipid bilayer membrane as a supporting structure limits their lifetime and the voltages that can be applied, thus restricting some applications. On the other hand, solid-state nanopores present increased durability over a wider range of experimental conditions, such as applied voltages, temperature and pH, and their size is tuneable in situ. In principle, solid-state nanopores offer a greater propensity to be integrated into robust lab-on-a-chip devices as arrays. In fact, recent studies revealed various integration strategies, which embed such nanopores within microfluidic networks. The nanopores used in these investigations are typically constructed in an ultrathin (10-nm to 50-nm) dielectric membrane (e.g. SiN) using high-energy ion or electron beams. However, the use of FIB or TEM to fabricate nanopores introduces integration challenges. The need for direct line-of-sight access when drilling with beams of energetic particles demands that nanopores be fabricated before their integration within microfluidic devices. This imposes strict alignment requirements during both nanopore fabrication and device assembly, resulting in challenges that limit the yield of functional devices, particularly for array formation on a single membrane or when the dimensions of the microfluidic channels are reduced in order to minimize electrical noise. More generally, these conventional nanofabrication techniques rely on the production of nanopores in a vacuum environment, which inevitably introduces handling risks and wetting issues when transitioning into aqueous solutions for biosensing experiments.
An alternative method of fabricating solid-state nanopores reliably using high electric fields was recently proposed and is referred to herein as nanopore fabrication by controlled breakdown (CBD). In situ and under typical experimental biological sensing conditions (e.g. in 1 M KCl), a dielectric breakdown event is induced in the supporting intact insulating membrane resulting in the formation of a single nanopore with a diameter as small as 1-nm in size but tuneable to large sizes with sub-nm precision. The simplicity of the CBD method lends itself well to the integration of nanopore sensors within complex microfluidic architectures and to potential lab-on-a-chip devices. Combining the advanced sample handling and processing capabilities inherent in microfluidic devices with in situ nanopore fabrication is expected to mitigate various integration issues and expand the range of applications of the sensing platform. Further details regarding this fabrication technique can be found in U.S. Patent Publication No. 2015/0108808 which is entitled “Fabrication of Nanopores using High Electric Fields” and is incorporated by reference herein in its entirety.
This section provides background information related to the present disclosure, which is not necessarily prior art.